The RNA-binding region of human TRBP interacts with microRNA precursors through two independent domains

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through RNA interference. Human miRNAs are generated through a series of enzymatic processing steps. The precursor miRNA (pre-miRNA) is recognized and cleaved by a complex containing Dicer and several non-catalytic accessory proteins. HIV TAR element binding protein (TRBP) is a constituent of the Dicer complex, which augments complex stability and potentially functions in substrate recognition and product transfer to the RNA-induced silencing complex. Here we have analysed the interaction between the RNA-binding region of TRBP and an oncogenic human miRNA, miR-155, at different stages in the biogenesis pathway. We show that the region of TRBP that binds immature miRNAs comprises two independent double-stranded RNA-binding domains connected by a 60-residue flexible linker. No evidence of contact between the two double-stranded RNA-binding domains was observed either in the apo- or RNA-bound state. We establish that the RNA-binding region of TRBP interacts with both pre-miR-155 and the miR-155/miR-155* duplex through the same binding surfaces and with similar affinities, and that two protein molecules can simultaneously interact with each immature miRNA. These data suggest that TRBP could play a role before and after processing of pre-miRNAs by Dicer.

Figure 1.

Analysis of the solution behaviour of dsRBDs from TRBP. (a) Diagram of the three constructs of TRBP used in this study. The same colour scheme for TRBP-D1 (red) and TRBP-D2 (green) is used throughout; (b) analytical SEC profiles showing elution volume vs. absorbance at 280 nm, A₂₈₀, for TRBP-D1 (red), TRBP-D2 (green) and TRBP-D12 (black). The molecular weight of each species determined by MALLS is given; (c) site-by-site comparison of the (¹H,¹⁵N) chemical shift of a residue in the single domain and the corresponding residue in TRBP-D12. Circles denote (¹⁵N) chemical shifts and squares (¹H) chemical shifts. Amide groups from TRBP-D1 are coloured in red and those from TRBP-D2 in green. The points corresponding to the large outliers are annotated in grey. RMSD values for ¹H and ¹⁵N chemical shifts are provided.

Figure 2.

NMR analysis of the interaction between TRBP-D12 and pre-miR-155. (a) Compound chemical shift values (Δδ) measured when TRBP-D12 interacts with pre-miR-155 plotted as a function of residue number (black). Δδ = [ + (Δδ_N/6.5)²]^½, where Δδ_X is the difference in chemical shift between apo- and RNA-bound spectra (39). Prolines and unassigned residues have been given values of −0.05 and −0.1 ppm, respectively. For comparison, the change in accessible surface area (ΔASA) when TRBP-D2 forms a complex with dsRNA is shown (green). ΔASA were calculated using the POPS* server (46) from 3ADL (18). The boundaries of secondary structure elements were taken from the 3D structures of TRBP-D1 [3LLH; (31)] and TRBP-D2 [3ADL; (18)]; (b,c) TOP: cartoon representation of the 3D structures of TRBP-D1 and TRBP-D2 showing the three regions in canonical dsRBDs that are implicated in dsRNA binding; (b,c) BOTTOM: Δδ values plotted on the respective 3D structures. Each amide nitrogen is represented by a sphere and coloured according to the Δδ scale provided. The β_2/3 loop was not resolved in the 3D structure of TRBP-D1 and is shown by a broken line.

Figure 3.

Comparison of NMR data collected of single- and double-domain constructs in complex with pre-miR-155. Two dimensional (¹H,¹⁵N) spectra of (a) TRBP-D1/pre-miR-155 in red, (b) TRBP-D2/pre-miR-155 in green, and (c) TRBP-D12/pre-miR-155 in black. A coloured graphical representation of each construct is provided above the spectrum. (d) Superposition of the spectra shown in (a-c) retaining the same colour scheme and construct graphics. (e) Comparison of the (¹H,¹⁵N) resonance frequencies of a residue in the single domain and the corresponding residue in TRBP-D12. Circles denote (¹⁵N) chemical shifts and squares (¹H) chemical shifts. Amide groups from TRBP-D1 are coloured in red and those from TRBP-D2 in green. The points corresponding to the large outliers are annotated. RMSD values for ¹H and ¹⁵N chemical shifts are provided.

Figure 4.

Characterization of the interaction between TRBP-D12 and pre-miR-155. (a) An example of ITC data recorded of the titration of pre-miR-155 with TRBP-D12. The top panel shows power vs. time together with a schematic of the components of the sample. The bottom panel shows the integrated injection enthalpy per mole of injectant plotted as a function of the ratio of protein and RNA concentrations. The data were fitted to a two-site sequential binding model shown by a solid black line. The two dissociation constants are given. The thermodynamic parameters calculated are provided in Supplementary Table S2. (b) Comparison of c(s) distributions of TRBP-D12 (grey) and pre-miR-155 (purple); (c) c(s) distributions of a sample prepared with 10 µM TRBP-D12 and different concentrations of pre-miR-155. The protein and/or RNA concentration corresponding to each curve is provided.

Figure 5.

Interaction of the RNA-binding region of TRBP with the substrate and product of Dicer. (a) An example of an ITC analysis of the titration of miR-155/miR-155* with TRBP-D12 showing power vs. time data (top, with a schematic of the components of the sample) and the integrated injection enthalpy per mole of injectant plotted as a function of the protein/RNA ratio (bottom). The data were fitted to a two-step sequential binding model shown by a solid black line. The two dissociation constants and their standard deviations are given. The complete thermodynamic parameters derived are provided in Supplementary Table S2. (b) Comparison of the changes in (¹H,¹⁵N) NMR spectra of TRBP-D12 resulting from the addition of either pre-miR-155 (left) or miR-155/miR-155* (right). The nucleotide sequence and predicted secondary structure of each RNA are provided. (¹H,¹⁵N) assignments of cross-peaks in each spectrum are annotated and coloured according to the dsRBD in which they reside (red, dsRBD-1; green, dsRBD-2).